The leaves of higher plants contain tightly-regulated openings called stomatal pores. The stomatal pores are located in the epidermis of plant leaves and are created by pairs of so-called “guard cells” which surround the actual opening. Guard cells control both the influx of CO2 as a raw material for photosynthesis and water loss from plants through transpiration to the atmosphere. The exact molecular mechanism underlying the regulation of the pore size is complex and not completely understood. However, the plant plasma membrane H+-ATPase (PMA) plays a pivotal role in this process. In particular, it is responsible for creating and maintaining an electrochemical proton gradient across the plasma membrane of guard cells that provides the driving force for nutrient uptake and maintenance of cell turgor. An increase in the proton gradient is known to result in osmotic swelling of the guard cells, consequently leading to an opening of the stomatal pore.
The polypeptide chain of the plasma membrane H+-ATPase has been shown to form ten transmembrane helices, the N-and C-terminal amino acids of which are both located at the cytoplasmic face of the plasma membrane. In addition, PMA appears to contain a C-terminal regulatory domain (Palmgren et al., 1991) which can act as an intrinsic inhibitor of the proton pump. The autoinhibitory activity of this regulatory domain is relieved by phosphorylation of the penultimate threonine residue and subsequent association with 14-3-3 proteins, as shown recently (Svennelid et al., 1999; Fugisang et al., 1999; Maudoux et al., 2000).
This interaction results in an increased proton pump activity, a swelling of guard cells and ultimately in an opening of stomatal pores. Depending on the degree of proton pump activity, the supply of nutrients and other factors such as ambient temperature, the plant will either show an increased growth rate or a massive loss of water. Several naturally occurring compounds are known to stabilize the interaction of 14-3-3 and PMA. One such compound is Fusicoccin (FC), a diterpene glycoside produced by the fungus Fusicoccum amygdali (Ballio et al., 1964). Despite the fact that the, fungus is host specific, FC exerts its effects in virtually any higher plant (Marre et al., 1979). Recently it has been shown that 14-3-3 proteins associate with the plant plasma membrane H+-ATPase to generate a ligand binding complex for Fusicoccin (Baunsgaard et al., 1998). However, neither the molecular interactions underlying ligand binding nor the nature of the binding pocket for Fusicoccin are known.
A better understanding of the nature of the ligand binding pocket created by the polypeptide chains of PMA and 14-3-3 in the presence of Fusicoccin would allow to identify the molecular interactions that are required to stabilize PMA in its active state. This could lead to the development of ligands and of transgenic plants with modified properties and would ultimately allow to adapt plants to adverse environmental conditions. Moreover, transgenic plants could be developed, encoding. mutant 14-3-3 or mutant PMA, which would be resistant to fusicoccin action. Such plants could be grown in the presence of fusicoccin, since the mutation would guarantee the selective survival of the transgenic plant. This would open the way to the development of a new class of herbicides to be used in conjunction with the transgenic plant. However, the study of ligand binding requirements of PMa14-3-3 or of the interactions of Fusicoccin is hampered by the fact that the spatial structure of the ligand binding pocket is still unknown. This is partly because crystallization of the ternary complex of PMA/14-3-3 and Fusicoccin has been unsuccessful up to now.
Thus, the technical problem underlying the present invention was to provide the crystal structure of the binding site of Fusicoccin bound to PMA and 14-3-3. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.